CN102605052B - Fluorescence detection kit for detecting deafness susceptibility gene GJB2 235delC and application of fluorescence detection kit - Google Patents
Fluorescence detection kit for detecting deafness susceptibility gene GJB2 235delC and application of fluorescence detection kit Download PDFInfo
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- CN102605052B CN102605052B CN201210033109.6A CN201210033109A CN102605052B CN 102605052 B CN102605052 B CN 102605052B CN 201210033109 A CN201210033109 A CN 201210033109A CN 102605052 B CN102605052 B CN 102605052B
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Abstract
The invention discloses a fluorescence detection kit for detecting deafness susceptibility gene GJB2 235delC. The kit comprises a pre-amplification reagent and a post-amplification reagent; the pre-amplification reagent comprises a reaction mixture comprising a PCR (Polymerase Chain Reaction) buffer solution, MgCl2 and dNTPs (deoxyribonucleotide Triphosphates), a Taq enzyme, ultrapure water and a primer mixture comprising high specificity amplification GJB2 235delC and an Amelogenin gene locus; and the post-amplification reagent comprises a genotyping standard substance and an interior label. According to the invention, a fluorescence labeling technology, a LNA (Locked Nucleic Acid) nucleoside monomer doping and primer modification technology and a capillary electrophoresis technology are firstly compounded to be adopted; the simultaneous detection on the high sensitivity specificity of the deafness gene locus GJB2 235delC is implemented; manpower and material resources and time are greatly saved; and the pollution caused by multi-step operation is prevented.
Description
Technical field
The present invention relates to the deaf sick tumor susceptibility gene GJB2 235delC fluorescence detection reagent kit of a kind of detection, belong to technical field of biological.
Background technology
The paathogenic factor research of hearing and speech handicapped person being carried out in world wide shows, about 60-80% patient's the cause of disease is relevant with inherited genetic factors, and wherein the clinical study data of developed country show, hereditary hearing impairment accounts for 80% in deafness patient.Therefore in recent ten years, the pathogenesis of hereditary hearing impairment and the research of molecular epidemiology thereof become one of most important content of deaf sick research.Along with the Human Genome Project completes, the location of deaf ospc gene and clone have obtained huge progress, and the molecule genetics research of deaf disease and the data of molecular epidemiology make investigators progressively recognize that deaf sick susceptibility gene mutation safeguarding that hearing is healthy and finding the importance of hearing in abnormal.
In the deaf-related gene of having located and having cloned at present, GJB2 is modal tumor susceptibility gene, in congenital severe deafness patient, accounts for 30-50%.At present, at this gene, had been found that 100 various mutations types, yet, the mutant form difference that not agnate GJB2 gene is common.In China, common mutant form is 235delC, and it accounts for 74.14% in all disease cause mutations, and approximately 22.2% Chinese non-syndromic hearing loss patient is relevant with this sudden change.Carry out before marriage and childbirth, antenatal, newborn infant GJB2 235delC deaf gene inspection, to instructing early stage and providing timely relief all significant.
Conventional gene tester has at present: direct Sequencing (direct sequencing, DS), Ligase detection reaction (ligase detection reaction, LDR), restriction fragment length polymorphism analysis (restriction fragment length polymorphism, RFLP), dhplc analysis (denaturing high performance liquid chromotography, DHPLC), quantitative PCR, gene chip.All there is different defects in these methods, such as complex operation, result, is difficult for the problems such as interpretation, poor repeatability, false negative false positive be many.
Adopt fluorescent mark technology, capillary electrophoresis technique, multiplex PCR composite amplification, by the multipair primer with different fluorescent markers simultaneously to the specific amplification of GJB2 235delC, and in these primers, mix nucleic acid analog to improve the Tm value of primer, further strengthen the specificity of primer, and go out peak situation by capillary electrophoresis post analysis PCR product, can realize the detection to this deaf gene site.The method is highly sensitive, interpretation is clear accurately, sampling is convenient.
Summary of the invention
The object of the invention is to provide a kind of deaf sick tumor susceptibility gene GJB2 235delC and Amelogenin locus fluorescence detection reagent kit of simultaneously detecting.
To achieve these goals, the technical scheme of taking: the deaf sick tumor susceptibility gene GJB2 235delC fluorescence detection reagent kit of a kind of detection, this test kit comprises the gene type reagent after amplifing reagent and amplification;
Before described amplification, reagent comprises: PCR damping fluid, MgCl
2, dNTPs mixture, Taq enzyme, the 5 pairs of primer mixtures and ultrapure water;
After described amplification, reagent comprises: interior mark ROX-300 and for genotyping standard thing corresponding to each gene mutation site of gene type;
The condition of using described test kit to carry out the reaction of PCR composite amplification: the pH value of pcr amplification system is 8.0-9.0, magnesium ion concentration is 1.5-3.5mM, the final concentration of 4 kinds of dNTP is respectively 200-300 μ M, the consumption of Taq enzyme is 0.1-0.4U/ μ L, and the list in 2 pairs of primer mixtures is 0.2-0.4 μ M to primer final concentration.
While using described test kit to carry out pcr amplification, amplification elementary reaction in a composite amplification reaction system, increase GJB2 235delC and Amelogenin locus simultaneously.
The primer detecting for GJB2 235delC is:
SEQ?NO.1：5’-CGCATTATGATCCTCGTTGTGG-3’
SEQ?NO.2：5’-GCCTGCAGCTGATCTTCGT-3’
Wherein LNA nucleosides represents with italics.
The primer detecting for Amelogenin locus is:
SEQ?NO.3：5’-CCCTGGGCTCTGTAAAGAAT-3’
SEQ?NO.4：5’-GCAGAGCTTAAACTGGGAAGC-3’。
2 pairs of described primers, wherein have 5 ' end of a primer to carry out fluorochrome label in every pair of primer, fluorescence dye used can be identical or different fluorescein.
Described composite amplification adopts polymerase chain reaction to realize, and adopts multiple tracks or single track capillary electrophoresis to detect.
The human gene group DNA that wherein detected is: use Chelex method, magnetic bead extraction method or phenol/chloroform extraction method to process to source sample the DNA obtaining; Described source sample is: derive from the mankind: filter paper blood cake/buccal swab sample, FTA card blood cake/buccal swab sample, buccal swab sample, blood/trace, tissue, amniotic fluid.
While using described test kit to carry out pcr amplification, amplification elementary reaction carries out on the PCR of any model instrument, amplification program: 94-98 ℃ 1-5min; 94-98 ℃ of 15-60s of 26-32 circulation, 55-65 ℃ of 15-60s, 72 ℃ of 15-60; 60 ℃ of 30-60min.
Use with fluorescent mark and through LNA nucleoside monomers, mix the primer of modification, improved the Tm value of primer, shortened primer length, thereby increased sensitivity and the specificity of primer, prevent false positive and false-negative appearance.
For detecting of Amelogenin, be on the one hand the effect of internal reference: whether indication template DNA is effectively or concentration range; Can effectively indicate whether to exist PCR product pollution on the other hand, prevent the false positive producing because polluting.
Accompanying drawing explanation
Fig. 1 is test kit of the present invention to the somatotype result after mutated individual, normal individual blood cake source DNA cloning; Somatotype result after not modified primer pair normal individual blood cake source DNA cloning.
Embodiment
Below in conjunction with the drawings and specific embodiments, the invention will be further described, but not as a limitation of the invention.
Embodiment 1
Deaf sick tumor susceptibility gene GJB2 235delC fluorescence detection reagent kit detects the DNA sample of sudden change and normal individual, the primer detecting for deaf sick tumor susceptibility gene GJB2 235delC adopts green fluorescence dyestuff HEX mark, primer for the amplification of Amelogenin locus adopts Yellow fluorochrome mark, and fluorescent marker is TAMRA; Interior mark adopts red fluorescence dyestuff mark, and fluorescent marker is ROX.
1, sample to be tested is 100 parts, and the technological method that all uses " DNA extraction-pcr amplification-order-checking " carried out order-checking to GJB2 235 sites and detects, and 5 parts, sample wherein suddenlys change.
2, the extracting genome DNA of sample
Chelex extraction method: cut 1～3mm blood cake and be placed in 1.5mL centrifuge tube, add sdH
2o 1mL, vibrates centrifugal, abandons supernatant liquor, and repeating step twice, abandons supernatant liquor, and after 5%Chelex-100 concussion is suspended, with the rifle head of cutting, draw 200 μ L fast and add in centrifuge tube, the vibration several seconds.After 56 ℃ of water bath heat preservation 30min, the vibration several seconds.95 ℃ of boiling water bath 10min, slightly vibrate the several seconds.The centrifugal 5min of 2000rpm, the DNA for extracting in supernatant.
3, the detection analysis of amplification and amplified production
3.1 pcr amplification systems:
3.2 pcr amplification programs:
4, amplified production fluoroscopic examination on genetic analyzer
By mark in deionized formamide and molecular weight, form loading mixture ((mark in 0.2 μ L molecular weight) * (sample introduction number)+(9.8 μ L deionized formamide) * (sample introduction number)).10 μ L loading mixtures are mixed with 1 μ L amplified production or alleles analysis standard substance, avoid producing bubble.95 ℃ of sex change 5min, ice bath 5min, and electrophoresis as early as possible.With genetic analyzer, detect and analyze.
5, conclusion
For example as shown in Figure 1:
The first row: confirmed cases, female, there is the detected peaks of peak value 1300H in interpretation position, GJB2 235delC site (237bp), and simultaneously another item chromosome normally goes out G2 peak (238bp, 1300H), and sex goes out X unimodal (105bp, 2400H);
The second row: confirmed cases, man, there is the detected peaks of peak value 1000H in interpretation position, GJB2 235delC site (237bp), simultaneously another item chromosome normally goes out G2 peak (238bp, 1100H), sex goes out X, Y bimodal (105bp, 1300H and 111bp, 1400H).
The third line: normal individual, man, interpretation position, GJB2 235delC site (237bp) does not go out the detected peaks of the existing peak value 2000H in ，JinG2 position, peak, and sex goes out X, Y bimodal (105bp, 800H and 111bp, 600H).
Fourth line: use not modified primer pair normal male individuality to carry out augmentation detection, interpretation position, GJB2 235delC site (237bp) is prone to small peak, if figure peak value is 900H, brings difficulty to interpretation.
Compare with sequence measurement: the present invention is consistent to the homologous genes seat somatotype result of the sample in same source; There is not false positive results.And use not modified primer pair normal male individuality to carry out augmentation detection, interpretation position, GJB2 235delC site is prone to small peak, occurs false positive, inconsistent with sequencing result, erroneous results.
2.5 * PCR damping fluid of different pH used in above embodiment, with the Tris-HCl damping fluid preparation of different pH values, the Tris-HCl concentration in 1 * PCR damping fluid is 10mM, KCl concentration is 50mM; In the present invention, Taq polysaccharase used and other reagent and material are commercially available prod.
Claims (2)
1. a deaf sick tumor susceptibility gene GJB2235delC fluorescence detection reagent kit, is characterized in that comprising archaeal dna polymerase and genotyping standard thing corresponding to the primer of buffered soln, amplification 235delC and Amelogenin locus, interior mark and each gene mutation site thereof;
The primer detecting for GJB2235delC is:
SEQ?NO.1：5’-CGCATTATGATCCTCGTTGTGG-3’
SEQ?NO.2：5’-GCCTGCAGCTGATCTTCGT-3’
Wherein LNA nucleosides represents with italics;
The primer detecting for Amelogenin locus is:
SEQ?NO.03：5’-CCCTGGGCTCTGTAAAGAAT-3’
SEQ?NO.04：5’-GCAGAGCTTAAACTGGGAAGC-3’；
The detection primer of described GJB2235delC and Amelogenin locus, has 5 ' end of a primer to carry out fluorochrome label in every pair of primer;
The composite amplification of described GJB2235delC and Amelogenin locus adopts polymerase chain reaction to realize, and adopts multiple tracks or single track capillary electrophoresis to detect.
2. test kit according to claim 1, is characterized in that: the primer that described GJB2235delC and Amelogenin locus detect can adopt identical or different fluorochrome label; Interior mark is selected and is different from above-mentioned fluorochrome label.
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| CN201210033109.6A CN102605052B (en) | 2012-02-14 | 2012-02-14 | Fluorescence detection kit for detecting deafness susceptibility gene GJB2 235delC and application of fluorescence detection kit |
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| CN1873027A (en) * | 2006-03-30 | 2006-12-06 | 韩东一 | Primer in use for in vitro diagnosing GJB2 mutation of deaf gene of autosomal recessive inheritance in non-syndrome |
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